Impurities in DNA: Purify the insert DNA. Ensure that any excess phenol, proteins, detergents, and ethanol from the DNA solution are removed.

Too much DNA was transformed: For chemically competent cells, add 1–10 ng of DNA in a volume of 5 µL or less per 100 µL of cells. For electrocompetent cells, add 10–50 ng of DNA in a volume of 1 µL or less per 20 µL of cells.

Although you will be picking white (recombinant) colonies, you should expect to see some blue (contain non-recombinant bacmid) colonies. Here are some possible causes for seeing no blue colonies and recommendations for the same:

We recommend analyzing your recombinant bacmid DNA by PCR analysis. Use the pUC/M13 Foward and Reverse primers that hybridze to sites flanking the mini-attTn7 site within the lacZalpha-complementation region to facilitate PCR analysis. Please see page 32 of the manual for further instructions.

High molecular weight bacmid DNA handled improperly: When isolating bacmid DNA, do not vortex the DNA solution; additionally, do not resuspend DNA pellets mechanically; allow solution to sit in the tube with occasional tapping.

This may be due to contamination or cytotoxicity from the bacmid prep. Make sure to include a negative control that is the bacmid only without Cellfectin® II Reagent. Additionally, use the PureLink® HiPure plasmid prep kit, not the silica-based miniprep kit for bacmid prep.

Mixture of Cellfectin® II Reagent and bacmid was not performed or was not incubated long enough: Mix the Cellfectin® II Reagent and bacmid well by tapping or gentle vortexing, and incubate the mixture for 15–45 min.

Bacmid yield is lower than estimated: Set up an optimization with different amounts of bacmid.

Bacmid is sheared during purification or freeze/thaw: Verify the integrity of bacmid on a gel.

Incubation time is not long enough: Incubate mix for 8 hr at 27°C.

Cells used are of high passages or have passed log-phase growth: For best results, use cells between 8–15 passages; plate cells when they are in log-phase growth.

Cellfectin® II Reagent has been frozen: Purchase a new vial.

Medium used contains serum: Use unsupplemented Grace’s medium in transfection.

Low transfection efficiency: We recommend using our Cellfectin® II Reagent for transfection; perform the transfection in Grace’s Medium, Unsupplemented and ensure no supplements, FBS, or antibiotics are present during the transfection. Harvest the viral supernatant when signs of infection are visible (typically >96 hr post-transfection).

Baculovirus is not recombinant: Verify transposition by PCR analysis of bacmid DNA using pUC/M13 forward and reverse primers; re-transfect insect cells with new recombinant bacmid DNA.

Use too low or too high viral titer: Vary the MOI.

Time of cell harvest is not optimal: perform a time course of expression to determine the optimal time to obtain maximal protein expression.

Cell growth conditions and medium are not optimal: Optimize cell culture conditions based on the size of your culture vessel and expression conditions; we recommend using Sf-900™ II SFM or Sf-900™ III SFM for optimal cell growth and protein expression.

Cell line is not optimal; try other insect cell lines.

Cells were harvested too late: Do a time-course experiment and harvest cells at different time points.

Viral stock was amplified using high MOI originally: Go back to the lower-passage viral stock and do a low-MOI amplification.

Did not spin down and get rid of cells when harvesting viral supernatant: Go back to the lower-passage viral stock and do a low-MOI amplification; if this viral stock is P2, this stock can be used in amplification.

For some genes, the virus can become very unstable: Free the aliquoted P2 viral stock and do one run of amplification after reviving.

Media used to culture insect cells usually have an acidic pH (6.0–6.5) or contain electron-donating groups that can prevent binding of the 6xHis-tagged protein to Ni-NTA. Amino acids such as glutamine, glycine, or histidine are present at significantly higher concentrations in media for growing insect cells than in media for growing mammalian cells, and compete with the 6xHis-tagged protein for binding sites on Ni-NTA matrices. Grace’s medium (Life Technologies), for example, contains approximately 10 mM glutamine, 10 mM glycine, and 15 mM histidine.

Dialysis of the medium against a buffer with the appropriate composition and pH (8.0) similar to the lysis buffer recommended for purification under native conditions usually restores optimal binding conditions. Note that depending on the medium used, a white precipitate (probably made up of insoluble salts) can occur, but normally the 6xHis-tagged protein remains in solution. This can be tested by either protein quantitation if using a protein-free medium or by monitoring the amount of 6xHis-tagged protein by western-blot analysis. After centrifugation, 6xHis-tagged protein can be directly purified from the cleared supernatant.

If you were using SF-900™ II SFM, that is incompatible with the ammonium acetate precipitation method. Our SF-900™ II Serum-Free Medium contains the block co-polymer non-ionic surfactant Pluronic F-68, which has been found to interfere with ammonium acetate precipitation. If you are putting the un-concentrated supernatant over the column, this should not cause a problem. If the supernatant has been concentrated, the Pluronic acid will need to be removed using a column.

Check the LR reaction by PCR analysis prior to transfection into insect cells.

We recommend using Grace’s Insect Cell Culture Medium, Unsupplemented during the transfection experiment instead of serum-free medium, as components in serum-free medium may interfere with transfection.

Ensure that FBS, supplements, or antibiotics are not included during transfection, as the proteins in these materials can interfere with the Cellfectin® II Reagent.

Use the LR recombination reaction using the pENTR/CAT plasmid as a positive control and Cellfectin® II Reagent only (mock transfection) as a negative control.

Ensure that cells are in the log phase of growth with >95% viability, and the amount of cells are in accordance with the suggestions in the manual.

Cells may not show signs of viral infection for up to a week depending on transfection efficiency; continue culturing and monitor cells daily for signs of infection.

Please check the construction of your entry clone, and ensure that the insert is in frame with the vector. Analyze the recombinant viral DNA by PCR to confirm the correct size and orientation of your insert after the LR reaction. Sequence your PCR product to verify the proper reading frame for expression of the epitope tag.

The incorrect MOI was used; ensure that the amount of viral stock was calculated correctly, and that an MOI of 5–10 was used. You may need to test a range of MOIs depending on the kinetics of expression of your recombinant protein.

The protein may be lost during cell lysis; if you are trying to detect an intracellular protein, analyze the supernatant to determine if the protein is being lost due to cell lysis.

Bac-N-Blue™ Expression System

Please compare your cells-only plate to the infected plate. The uninfected cells should appear overgrown when compared to infected cells, as transfection inhibits growth. If this is seen, keep checking the infected cells daily for other signs of infection (nuclear swelling, detachment from the plate, viral budding, and lysis). The kinetics of infection may be slower than expected. If cell growth does not appear to be inhibited, you may consider the following factors:

How was the DNA prepared? We recommend using a resin-based DNA purification system, such as our PureLink® HiPure Plasmid Prep Kits.

Were cells in log phase? What was their confluency? We recommend that cells be in log phase, 95% viable, and plated at 50–70% confluency for successful transfection.

What transfection reagent was used? We recommend using Cellfectin® II Reagent.

If there is contamination with wild-type virus in all of your samples, take a P1 viral stock and redo the plaque assay. Be sure to select well-spaced, occ– plaques. If you are having difficulty distinguishing a recombinant plaque from a non-recombinant plaque, try using one of the pBlueBac vectors. Recombinant plaques will be blue when chromogenic substrate is incorporated into the medium during the agarose overlay (see page 14 of the manual for more information).

Plaque Assay and Viral Stocks

The kinetics of infection may be slower than expected. Observe plates until the 8–9th day after infection. If no plaques appear, investigate the following:

If the cells are not healthy, then poor-quality or no plaques can result. Ideally, cells should be in mid-log phase and have a viability of greater than 90%. Cells should double at least once before infection stops growth. Ensure that the correct amount of cells was used at ~70% confluency.

The viral replication cycle can be inhibited due to poor nutritional and physical conditions of the cell.

The temperature of the agarose is also crucial. After overlaying the agarose, the plates should be left untouched for 1 hour for the agarose to completely solidify.

Excessive condensation during incubation at 27°C can inhibit plaque formation—remove paper towels or open the container containing plates as soon as condensation appears.

The viral titer is too low: Use a higher viral titer. You may need to re-infect your cells and collect a higher titer of your viral stock.

Yes, this is indicative of an aspirating problem on the plaques. The agarose overlays were "floating" because the medium was not completely aspirated from the plates. The plates need to be completely dry before the agarose is placed over the cells, especially when plaques will be picked. To do this, we typically tip the plate slightly and keep going around the rim of the plate with the Pasteur pipette tip, being careful not to disturb the cell monolayer. If any medium pooling at the rims of the plates (they will be small pools) is seen, continue to aspirate. This “floating" agarose overlay problem may also result in wild-type contamination. The wild-type virus is able to migrate to other portions of the plates and contaminate recombinant plaques. Wild-type virus replicates much faster than recombinant virus, and can quickly overwhelm the recombinant virus.

Cells are being singed when plated with hot melted agarose. This lyses the cells and releases lacZ into the agarose, turning it blue. Double-check plating temperatures. If plates are too wet, the blue can diffuse.

On the day you intend to pick plaques, make a solution of Bluo-gal in DMSO at 20 mg/mL. Add 50 µL per plate and spread with a glass spreader under sterile conditions. Wait 30–60 min, and your plaques should turn blue.

Yes, cells are infected with wild-type virus individually and will develop polyhedra at different rates until all the cells in the flask are infected. The polyhedra in cells will form in approximately 3–4 days, differing in size and number until they reach their maximum capacity and burst the cell, releasing tiny particles of virus into the medium.

This is typically an indication of poor homologous recombination. Check the plasmid/linear DNA ratio you used. If there are some blue plaques, however, expand those viruses and check for their protein. In our experience, they are correct, even if they were in relatively low abundance.

In the case of a blue colony, the E. coli has the bacmid and the plasmid in it, allowing the cells to survive the selection process. However, because the transposition has not occurred, the LacZ gene is not disrupted. For bulls-eye colonies, this indicates that the transposition took place when the colony was growing. Re-streaking for an isolated clone from the white portion of the mixed colony should yield some colonies where transposition occurred.